Lyophilised Formulation For Nucleic Acid Amplification And Testing
Abstract:
Methods for amplifying a target nucleic acid by self-sustained amplification methods are described. The methods are designed, in particular, to be carried out without use of specialised lab facilities or instruments. Compositions, lyophilised formulations, and kits for carrying out the methods are also described.
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This invention relates to methods for amplifying a target nucleic acid, and to methods for nucleic acid testing, particularly methods that can be carried out without specialised lab facilities or instruments. Compositions, formulations, and kits for nucleic acid amplification are also provided, as well as lyophilised formulations and kits comprising lyophilised formulations.
Nucleic acid testing is used for many purposes such as screening and diagnosis of infectious diseases and genetic disorders, testing for disease susceptibility, monitoring progression of treatment, and improving the safety of blood supplies. Nucleic acid testing combines the advantages of direct and highly sequence-specific detection of nucleic acid of an infectious agent with an analytic sensitivity that is several orders of magnitude higher than that of immuno-based tests, or virus isolation and cell culture methods. Nucleic acid testing also reduces the risk of infectious agent transmission between infection and seroconversion, of infection with immunovariant viruses, and of immunosilent or occult carriage.
Well known methods of nucleic acid testing involve use of reverse transcription of RNA followed by PCR (RT-PCR) to amplify RNA species. However, RT-PCR suffers from the disadvantage that it involves repeated wide changes in sample temperature, for which specialised thermal cycling instruments are required. A further disadvantage is that amplified RNA template can be difficult to differentiate from amplified contaminating double stranded DNA.
An alternative RNA amplification strategy to RT-PCR is termed transcription-based amplification. Such methods involve amplification of an RNA template using reverse transcriptase (RT), RNase H, and RNA polymerase activities, and include nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA), and self-sustained sequence replication (3SR) (Chan and Fox, Rev. Med. Microbiol. 10: 185-196 (1999); Guatelli et al., Proc. Natl. Acad. Sci. 87:1874-1878 (1990); Compton, Nature 350:91-92 (1991)). NASBA and 3SR use RT from Avian Myeloblastosis Virus (AMV) (which also has RNaseH activity), RNase H from Exoli, and T7 RNA polymerase. TMA uses Moloney Murine Leukemia Virus (MMLV) RT (which also has RNase H activity), and T7 RNA polymerase.
Transcription-based amplification methods have several advantages over RT-PCR. The reactions occur simultaneously in a single tube, and are carried out under isothermal conditions so a thermocycler is not required. The amplification reaction is faster than RT-PCR (lxlO'-fold amplification can be seen after five cycles, compared with lxl06-fold amplification after 20 cycles for RT-PCR). DNA background does not interfere with transcription-based amplification, and so these methods are not affected by double stranded DNA contamination. The amplification product is single stranded and can be detected without any requirement for strand separation.
Conventional transcription-based amplification methods, however, suffer from the disadvantage that they have lower specificity than RT-PCR. It is also necessary to denature the nucleic acid template by heating the sample, and then cooling before adding the enzymes required for amplification of the template, thereby increasing the complexity of the method. Transcription-based amplification methods are also not as robust as RT-PCR. Conventional NASBA is sensitive to temperature fluctuations exceeding +/- 0.5°C.
US 5,981,183 describes a method of transcription-based amplification which uses thermostable enzymes. The amplification reaction is carried out at 50-70°C, thereby improving specificity. However, a disadvantage of this method is that it is still susceptible to false positives caused by false priming of denatured double stranded DNA. Such methods are also not well suited to use in the field because of the need to heat the reaction to 50-70°C.
A further disadvantage of conventional nucleic acid testing is that detection of the amplified reaction product requires time-consuming, labour-intensive electrophoretic separation of the reaction products, or expensive equipment to detect fluorescent or chemiluminescent signals. The reagents required are expensive and must be transported and stored below room temperature. Separate designated areas are required for the sample preparation, amplification, and detection steps of the methods. The methods can only be carried out in specialized, well-equipped laboratories, by highly trained technicians. Consequently, conventional methods are not suitable for near-patient or field testing, and are unaffordable in poorer regions with a high prevalence of infectious disease (such as Africa, Asia, and Latin America) where they are most needed.
There is, therefore, a need to provide methods of nucleic acid testing that can be carried out without use of specialised lab facilities or instruments, and which have high specificity for target nucleic acid.
Lyophilisation has been used to store enzymes for nucleic acid amplification reactions. US 5,556,771 describes lyophilised formulations that comprise MMLV RT and T7 polymerase with trehalose and polyvinyl pyrrolidine as crypoprotectant stabilizing agents. However, the results described in US 5,556,771 show some loss in activity (measured as die ability to cause nucleic acid amplification) after storage at 35°C for 61 days. Instructions provided with commercially available kits comprising lyophilised reagents for carrying out transcription-mediated amplification (GEN-PROBE® APTIMA® General Purpose Reagents (GPR) 250 Kit) or PCR amplification (SmartMix™ HM of Cepheid) require the lyophilised reagents to be stored at 2-8°C. Instructions provided with a commercially available kit containing lyophilised reagents for NASBA-based nucleic acid amplification (Nuclisens® Basic Kit Amplification Reagents of Biom6rieux) specifies that the amplification reagents should be stored at <-20°C. We have also found that lyophilised formulations disclosed in US 5,556,771 and in the above commercially available kits do not reconstitute rapidly, but instead require extensive mixing in specially formulated reconstitution buffers.
There is, therefore, a need to provide lyophilised formulations that can preserve labile reagents in a stable condition for long periods at ambient temperature, and which can be easily and rapidly rehydrated.
According to the invention there is provided a method of nucleic acid amplification, which comprises amplifying a target nucleic acid by a self-sustained amplification reaction which is carried out at a temperature between 42°C and 50°C.
The term "self-sustained amplification reaction" is used herein to include nucleic acid amplification reactions in which copies of a target nucleic acid are produced, which then function as templates for production of further copies of target nucleic acid (either sense or anti-sense copies). The reactions are self-sustained reactions that can occur under isothermal conditions, and so there is no requirement for thermal cycling during the amplification reaction (unlike, for example, the polymerase chain reaction (PCR)). Examples of self-sustained amplification reactions are known to the skilled person, and include transcription-based amplifications, strand displacement amplification (SDA), rolling-circle amplification (RCA), Q beta replicase amplification, and loop-mediated isothermal amplification (LAMP).
It has been found that the time taken to obtain a desired copy number of amplification product using a method of the invention is surprisingly much less than the time taken to achieve the same copy number with conventional self-sustained amplification methods. In our experience,
methods of the invention are approximately twice as quick as conventional methods. A suitable incubation time at between 42°C and 50°C is at least 30 minutes, or at least 40 minutes. 45-55 minutes may be optimal.
"Whilst the temperature may vary between 42°C and 50°C when carrying out the amplification reaction, it is expected that the amplification reaction will generally be carried out under substantially isothermal conditions, i.e. within a temperature range of 1-3°C. A suitable way of achieving this is to incubate the amplification reaction using a water bath.
The phrase "at a temperature between 42°C and 50°C" means above 42°C and less than 50°C. The self-sustained amplification reaction may be carried out at a temperature above 43°C and less than 50°C. The self-sustained amplification reaction may be carried out at a temperature of 43-49°C. The reaction may be carried out at a temperature of 43-49°C, 44-49°C, 45-49°C, or45-48°C.
The target nucleic acid may be DNA (single or double stranded) or RNA. The target nucleic acid may be any target nucleic acid that it is desired to amplify or detect, including ribosomal RNA, viral or bacterial RNA or DNA. The target nucleic acid may be nucleic acid of (or derived from) a disease causing micro-organism (for example HCV). The target nucleic acid may be nucleic acid of an organism associated with a sexually transmitted disease, such as Chlamydia trachomatis, or HTV. In other embodiments, the target nucleic acid may be nucleic acid of a subject (for example to determine a particular genotype of the subject).
Methods of the invention may be used to determine whether or not a target nucleic acid is present in a sample solution suspected of containing the target nucleic acid. Accordingly, there is also provided according to the invention a method of testing for the presence of a target nucleic acid in a sample solution suspected of containing the target nucleic acid, the method comprising incubating the sample solution under conditions for amplification of the target nucleic acid by a self-sustained amplification reaction at a temperature between 42°C and 50°C.
The sample solution may be any solution suspected of containing a target nucleic acid which it is desired to detect. The sample solution may be, or be derived from, a biological sample obtained from a subject. Examples of biological samples are blood, serum, urine, a cervical smear, a swab sample, or tissue homogenate.
It will be appreciated that it may be necessary to extract nucleic acid from the biological sample to provide a sample solution that can be tested in accordance with the invention to determine whether or not a target nucleic acid is present. Nucleic acid extraction may be carried out using any suitable nucleic acid extraction method. Suitable methods include solid phase extraction of nucleic acid on silica particles (Boom et al., J. Clin. Microbiol; 28:495-503, 1990) or silica gel/glass fibre filters (Vogelstein et al., Proa-Natl. Acad. Sci., 76: 615-619,1979) in the presence of chaotropic salts, using commercially available-kits (for example from Qiagen, Roche, or Invitrogen). Alternative methods include: liquid phase extraction technology based in acid guanidinium thiocyanate-phenol-chloroform extraction (Chomczynski et al., Anal Biochemistry; 162: 156-159, 1987); FTA® kit protocol by Whatmann using FTA® paper filter matrixes impregnated by chemical formula that lyses cell membranes and immobilizes the nucleic acids; Charge Switch™ technology (Baker et al., EP1036082). Alternative, more simple procedures involve sample lysis by heat or chemical treatment and sample dilution prior to amplification.
Methods of the invention for determining whether or not a target nucleic acid is present in a sample solution may be used to test a biological sample obtained from a subject to see whether the subject is infected with an infectious agent, or to monitor the subject for progression of a disease, or for response to treatment.
Examples of infectious agents include bacterial or viral infectious agents, such as HIV, HCV, HPV, CMV, HTLV, EBV, rhinovirus, measles virus. Infectious agents may be those associated with sexually transmitted disease, such as Chlamydia trachomatis, or HTV.
Suitable self-sustained nucleic acid amplification reactions may be carried out using the following enzyme activities: an RNA-dependent DNA polymerase, a DNA-dependent DNA polymerase, a DNA/RNA duplex-specific ribonuclease, and a DNA-dependent RNA polymerase.
The RNA-dependent DNA polymerase, DNA-dependent DNA polymerase, and DNA/RNA duplex-specific ribonuclease activities may be provided by a single enzyme (for example AMV-RT or MMLV-RT). Additional DNA/RNA duplex-specific ribonuclease activity may be provided by a separate enzyme (for example an RNaseH). In some embodiments, AMV-RT and RNaseH may be used (as in NASBA).
As explained above, self-sustained nucleic acid amplification reactions are known to the skilled person. Suitable types of reaction that may be used in accordance with the invention
include transcription-based amplification methods, such as methods corresponding to NASBA, TMA, or 3SR (i.e. methods which are the same as conventional NASBA, TMA, or 3SR, but carried out at a temperature between 42°C and 50°C).
A transcription-based self-sustained amplification reaction suitable for use in methods of the invention is described below, with reference to Figure 1.
An antisense Primer 1 comprises nucleic acid sequence complementary to a portion of a target RNA so that the primer can hybridise specifically to the target RNA, and a single stranded-version of a promoter sequence for a DNA-dependent RNA polymerase at its 5'-end. Primer 1 is annealed to the RNA target. An RNA-dependent DNA polymerase extends Primer 1 to synthesise a complementary DNA (cDNA) copy of the RNA target. A DNA/RNA duplex-specific ribonuclease digests the RNA of the RNA-cDNA hybrid. A sense Primer 2 comprises nucleic acid sequence complementary to a portion of the cDNA. Primer 2 is annealed to the cDNA downstream of the part of the cDNA formed by Primer 1. Primer 2 is extended by a DNA-dependent DNA polymerase to produce a second DNA strand which extends through the DNA-dependent RNA polymerase promoter sequence at one end (thereby forming a double stranded promoter). This promoter is used by a DNA-dependent RNA polymerase to synthesise a large number of RNAs complementary to the original target sequence. These RNA products then function as templates for a cyclic phase of the reaction, but with the primer annealing steps reversed, i.e., Primer 2 followed by Primer 1.
In a variation of this method, Primer 2 may also include a single stranded version of a promoter sequence for the DNA-dependent RNA polymerase. This results in production of RNAs with the same sense as the original target sequence (as well as RNAs complementary to the original target sequence).
In some conventional self-sustained transcription-based amplification reactions it is known to cleave the target RNA at the 5'-end before it serves as the template for cDNA synthesis. An enzyme with RNase H activity is used to cleave the RNA portion of an RNA-DNA hybrid formed by adding an oligonucleotide (a cleavage oligonucleotide) having a sequence complementary to the region overlapping and adjacent to the 5'-end of the target RNA. The cleavage oligonucleotide may have its 3'-terminal-OH appropriately modified to prevent extension reaction. Whilst in some embodiments of the invention a cleavage oligonucleotide could be used, it is preferred that a method of the invention is carried out in the absence of a cleavage oligonucleotide thereby simplifying the amplification reaction and the components required.
It will be appreciated that in addition to the required enzyme activities, it will also be necessary to provide appropriate nucleotide triphosphates (for transcription-based amplifications ribonucleotide triphosphates (rNTPs, i.e. rATP, rGTP, rCTP, and rUTP), and deoxyribonucleotide triphosphates (dNTPs, i.e. dATP, dGTP, dCTP, and dTTP) are required), appropriate primers for specific amplification of the target nucleic acid, a suitable buffer for carrying out the amplification reaction, and any necessary cofactors (for example magnesium ions) required by the enzyme activities. Examples of suitable buffers include Tris-HCl, HEPES, or acetate buffer.
Accordingly, conditions for amplification of the target nucleic acid used in methods of the invention for testing for the presence of a target nucleic acid in a sample solution may comprise enzyme activities required for the self-sustained amplification reaction (for example, RNA-dependent DNA polymerase, DNA-dependent DNA polymerase, DNA/RNA duplex-specific ribonuclease, and DNA-dependent RNA polymerase enzyme activities), cofactors required by the enzyme activities (for example magnesium ions), primers for specific amplification of the target nucleic acid, appropriate nucleotide triphosphates (ribonucleotide triphosphates and deoxyribonucleotide triphosphates are required for transcription-based amplifications). Conditions for amplification of the target nucleic acid should also include a suitable buffer (such as Tris-HCl, HEPES, or acetate buffer). A suitable salt may be provided, such as potassium chloride or sodium chloride.
Suitable concentrations of these components may readily be determined by the skilled person. We have found that suitable rNTP concentrations are typically in the range 0.25-5mM, preferably 0.5-2.5mM. Suitable dNTP concentrations are typically in the range 0.25-5mM dNTP, preferably 0.5-2.5mM. Suitable magnesium ion concentrations are typically in the range 5-15mM.
Whilst it is possible that the self-sustained amplification reaction may be carried out in the temperature range of between 42°C and 50°C using thermostable enzymes, the Applicant has appreciated that non-thermostable enzymes may be used in this temperature range (provided the non-thermostable enzyme retains activity between 42 and 50°C). The term "thermostable enzyme" is used herein to mean an enzyme with optimal enzymatic activity at 50°C or above. Typically a. thermostable enzyme maintains its activity at temperatures at least in excess of 55°C and up to about 72°C or higher. A "non-thermostable enzyme" has optimal enzymatic activity below 50°C, suitably in the temperature range of 37-41°C (although the non-thermostable enzyme may still retain activity at 50°C or above). Accordingly, in certain
embodiments of the invention, at least one of the enzyme activities (for example the DNA-dependent RNA polymerase activity) is provided by a non-thermostable enzyme. In some embodiments, all of the enzyme activities use for the amplification reaction may be provided by non-thermostable enzymes. Use of non-thermostable enzymes is preferred because this allows the amplification reaction to proceed efficiently at a temperature between 42°C and 50°C. Thermostable enzymes generally have optimum activity at temperatures above this range.
Some conventional transcription-based amplification methods use very high amounts of T7 RNA polymerase (for example 142 or more units, where one unit incorporates Inmole of labelled nucleotide into acid insoluble material in 1 hour at 37°C under standard assay conditions, such as: 40mM Tris-HCl (pH8.0), 50mM NaCl, 8mM MgCfc, 5mM DTT, 400uM rNTPs, 400|xM [3H]^UTP(30cptn/pmoles), 20n,g/ml T7 DNA, 50^g/rol BSA, lOOui reaction volume, 37°C, lOmin.). We have found that methods of the invention can be carried out using significantly less T7 RNA polymerase than such conventional methods, thereby reducing cost. Thus, methods of the invention are preferably carried out using less than 142 units of a DNA-dependent RNA polymerase (for example T7 RNA polymerase), suitably less than 100 units or less than 50 units, such as 30-40 units.
It may be desirable to include one or more agents that may facilitate or enhance the self-sustained amplification reaction at temperatures between 42°C and 50°C. An agent may facilitate or enhance the reaction by any mechanism, but typically the agent is not essential for the reaction to take place, and may not directly take part in the reaction. Some such agents may act by helping to stabilize the activity of an enzyme required for the self-sustained amplification reaction between 42°C and 50°C, or by reducing the effect of any inhibitors of the self-sustained amplification reaction that may be present.
Examples of suitable agents include the following:
i) an inert protein. The term "inert protein" is used herein to mean a protein which
does not take part in the amplification reaction. Suitable examples include bovine serum^ albumin (BSA), casein, gelatin, or lysozyme. A suitable concentration range of the inert protein is 0.01-lu,g/|il. The protein should be RNase-free;
ii) a reducing agent, such as Dithiothreitol (DTT) (for example at a concentration of l-5mM) or n-acetylcysteine (NAC) (for example at a concentration of 0.1-1M);
iii) an inert amphophilic polymer (which is not a protein). The term "inert amphiphilic polymer" is used herein to mean an amphiphilic polymer which does not take part in the amplification reaction. The inert amphiphilic polymer may be non-charged. Examples of inert amphipihlic polymers are polyvinyl pyrrolidone (PVP) and polyethylene glycol (PEG) or other similar polyole. A suitable concentration range of the inert amphiphilic polymer is 0.1-2%;
iv) a sugar-alcohol, for example sorbitol, mannitol, or glycerol (for example up to 5% glycerol). A suitable concentration range is 0.1-2 M;
v) a low molecular weight saccharide, suitably a monosaccharide, disaccharide, or trisaccharide. Examples of disaccharides are trehalose, sucrose and maltose. A suitable concentration range is 2.5%-15%;
vi) homopolymeric nucleic acid (200-2000 bases). Examples include: Poly A, Poly C, or Poly U nucleic acid (for example at 100-600 ng/amplification); poly dA, poly dC or poly dl (for example at 100-600 ng/amplification); tRNA; rRNA;
vii) acetate salts, for example magnesium or potassium acetate;
viii) spermidine, for example at 0.5-3mM;
ix) poly-Lysine, for example at 0.2-3mM;
x) detergent, for example NP40 or Tween20 (suitably at 0.01-0.5%).
The concentration of each of the above agents may be optimised for each different target nucleic acid and set of primers used for amplification.
Other agents may act by facilitating primer annealing or facilitating denaturation of double stranded nucleic acid. Accordingly, it may be desired alternatively or additionally to include in the amplification reaction one or more agents that facilitate primer annealing and/or one or more agents mat facilitate denaturation of double stranded nucleic acid.
Examples of agents that facilitate primer annealing are positively charged ions, such as potassium or sodium ions. Potassium ion may be provided by potassium chloride or acetate (suitably at a concentration of 30-200mM or 30-90mM). Sodium ion may be provided by sodium chloride or acetate (suitably at a concentration of 50-400mM).
Examples of agents that facilitate denaturation of double stranded nucleic acid are:
i) polar aprotic solvents, such as dimethyl sulfoxide (DMSO), for example at a
concentration of 3-20% (v/v), or less than 10% (v/v). It has been found that when nucleic acid amplification is carried out in the temperature range of between 42 and 50°C, the amount of polar aprotic solvent required to have an effect is less than the amount required at lower
temperatures. Alternative polar aprotic solvents that may be used include tetramethylene sulfone, or tetramethylene sulfoxide;
ii) a zwitterionic compound, such as betaine (N, N, N-trimethylglycine), for example at a concentration of 0.2-3M. Betaine may be used in place of DMSO. An advantage of betaine is that it is more stable than DMSO, and it does not appear to inhibit lyophilisation. Consequently, use of betaine instead of DMSO may be suitable where lyophilised reagents are used for the amplification reaction. In some circumstances, however, use of betaine and DMSO may be desired since synergistic effects of these reagents on double stranded nucleic acid denaturation have been observed. Alternative zwitterionic compounds that may be used include monomethylglycine, dimethylglycine, D-carnitine, homoectoine, L-ectoine or derivatives;
iii) a modified nucleotide triphosphate (NTP) that comprises a base that can base pair with guanine or cytosine such that the melting temperature of the base pair is less than the melting temperature of a guanine-cytosine base pair. An example of a modified NTP is inosine triphosphate (rITP). An inosine-cytosine (I-C) base pair comprises two hydrogen bonds between the bases, compared with a guanine-cytosine (G-C) base pair which comprises three hydrogen bonds, and so the melting temperature of the I-C base pair is less than a G-C base pair. For example, rITP may be present in the amplification reaction at a concentration of 0.5-4mM or 0.5-3.5mM. When rITP is present, the amount of rGTP in the amplification reaction may be reduced compared to the amounts of the other ribonucleotide triphosphates (rATP, rCTP, rUTP) to compensate for the amount of rITP. The ratio of rITPrrGTP will depend on the target nucleic acid and on the primers that are used. A typical ratio is 1:3 to 1:1.5 rITP:rGTP;
iv) a single-stranded nucleic acid binding protein, such as Single-stranded Binding Protein (SSB), or Phage T4 gene 32 protein. SSB and Phage T4 gene 32 protein bind single stranded regions of DNA and thereby inhibit formation of double stranded DNA or DNA/RNA hybrids.
Other agents may improve the specificity of the amplification reaction. Accordingly, it may be desired alternatively or additionally to include one or more such agents in the amplification reaction.
Examples of agents that may improve the specificity of the amplification reaction are:
i) ammonium ions, such as tetramethylammonium chloride (TMAC). Ammonium ions
are thought to interfere with week hydrogen bond formation and create more stringent and specific hybridisation conditions;
ii) EDTA, EGTA, nitrioacetic acid (NTA), uramil diacetic acid (UDA), trans-1,2-
cyclohexanediaminetetraacetic acid (CyDTA), diethylenetriaminepentaacetic acid (DPTA),
elliyleneglycolbis(2-aminoethyl)eraer diarninetetraacetic acid (GEDTA),
triethylenetetraminehexaacetic acid (TTHA), or a salt thereof. These compounds are believed to improve the signal-to-noise ratio of amplification reactions by significantly inhibiting the occurrence of non-specific amplification reactions;
iii) carrier nucleic acid with one or more magnesium salts. This is believed to reduce polymerase extension of non-target nucleic acids during amplification through a reduction in the amount of primer-dimer formation.
In conventional transcription-based amplification methods, it is generally necessary to carry out an initial incubation of sample containing the target nucleic acid at a raised temperature before cooling the sample. This reduces secondary structure in the target nucleic acid, and allows primers for use in the amplification reaction to anneal to the target nucleic acid. Unless enzymes are used that retain activity after incubation at the raised temperature, this initial incubation step is carried out before addition of the enzymes required for the amplification reaction.
It has been found, however, that carrying out the self-sustained amplification reaction at a temperature between 42°C and 50°C does not require a preliminary raised temperature incubation step to be carried out. Consequently, methods of the invention are simplified compared to conventional transcription-based amplification methods, and can be carried out more rapidly than such methods. These are particularly important advantages for nucleic acid amplification reactions carried out in the field.
Of course, in some circumstances, it may nonetheless be desirable to carry out a raised temperature pre-incubation step. If the target nucleic acid is double stranded DNA, it will usually be necessary to denature the target before carrying out the amplification reaction. Double stranded DNA may be denatured by chemical methods well known to the skilled person, or alternatively by raised temperature denaturation.
Once the amplification reaction has been carried out, it will usually be desired to detect product of the amplification reaction (referred to as "amplification product" below). Single or double stranded amplification product may be detected. For example, double stranded amplification product produced during the cyclic phase of the self-sustained amplification reaction illustrated in Figure 1 and described above may be detected. If single stranded
amplification product is detected this removes any requirement for separation of double strands, and therefore simplifies detection.
Detection of amplification product may be carried out using any suitable method. For example, an instrument-independent detection method may be used, for example allowing visual detection of the amplification product.
According to particular embodiments of the invention amplification product may be detected using a dipstick. In suitable methods of dipstick detection, amplification product is transported along a dipstick by capillary action to a capture zone of the dipstick, and detected at the capture zone. Amplification product may be captured and detected using a sandwich nucleic acid dipstick detection assay in which the amplification product is immobilised at the capture zone of the dipstick by hybridisation to a capture probe, and detected at the capture zone by hybridisation to a detection probe.
Methods of detection of nucleic acid by dipstick assay are known to the skilled person. The Applicant has developed particularly sensitive methods of dipstick detection, which are described in WO 02/004667, WO 02/04668, WO 02/004669, WO 02/04671, and in Dineva et al (Journal of Clinical Microbiology, 2005, Vol. 43(8): 4015-4021).
It.is well known that a disadvantage of conventional nucleic acid amplification reactions is the risk of contamination of target nucleic acid with non-target nucleic acid that can lead to false positives. Conventionally, the risk of contamination in nucleic acid amplification reactions is minimised by carrying out the reactions in laboratories using separate dedicated areas for sample preparation, nucleic acid amplification, and detection of amplified nucleic acid. It will be appreciated, however, that this is not possible when nucleic acid amplification reactions are carried out away from such facilities (for example in the field, in a physician's office, at home, in remote areas, or in developing countries where specialist facilities may not be available).
The Applicant has appreciated that when a nucleic acid amplification reaction is carried out away from specialised lab facilities, risk of contamination can be reduced by performing the amplification reaction in a processing chamber that is sealed from the external environment. The nucleic acid amplification reaction may be carried out in accordance with a method of the invention. Detection of the amplification product may then be carried out in an analysing chamber that is also sealed from the external environment.
The processing chamber and analysing chamber may be provided by a device. The device may be preloaded with reagents (preferably in lyopbilised form) required for amplification of the target nucleic acid (including enzyme activities) and/or detection of the amplification product.
The risk of contamination of other samples with amplification product can be reduced by treatment of the amplification product with nucleic acid modifying or hydrolysing agents that prevent its further amplification. A suitable treatment is chemical treatment that modifies and degrades nucleic acid, for example non-enzymatic degradation of nucleic acid by chemical nucleases. Examples of chemical nucleases are divalent metal chelate complexes, such as copper Phenantroline-Cu (H) or Ascorbate-Cu (II) cleavage, as described by Sigman et al (LBiol. Chem (1979) 254, 12269-12272) and Chiou (J. Biochem (1984) 96, 1307-1310). Alternatively, a base that is not naturally present in the target nucleic acid can be incorporated into the amplification product. For example, dUTP can be used to incorporate uracil into a DNA amplification product (as described in US 5,035,996). If, prior to amplification, uracil DNA glycosylase (UDG) is then added to a sample that may have been contaminated with such DNA amplification product this will cause enzymatic hydrolysis of any contaminating amplification product (containing uracil) without affecting natural DNA in the sample.
Reagents required for amplification of the target nucleic acid and/or detection of the
amplification product may be provided in lyophilised form. Lyophilisation improves the
stability of the reagents, thereby allowing them to be stored for longer periods at higher
temperatures. Lyophilisation also reduces the weight and volume of the reagents so that they
are easier to transport. Use of lyophilised reagents is, therefore, advantageous for carrying out
> methods of the invention in the field.
The Applicant has developed lyophilisation formulations (i.e. formulations suitable for lyophilisation) which (once lyophilised) are able to maintain reagents in a stable condition at temperatures up to 37°C for at least a year. This removes any requirement for cold storage or cold-chain transport of the reagents. The formulations also have the advantage that they can be rapidly rehydrated after lyophilisation. This is a particularly desirable property of lyophilised formulations used for nucleic acid testing in the field since the speed or accuracy of a test can be adversely affected if a reagent required for amplification of a nucleic acid target or detection of amplification product is not rehydrated readily during the amplification or detection method.
According to the invention there is provided a lyophilisation formulation comprising a polysaccharide, a low molecular weight saccharide, and optionally a labile reagent which it is desired to preserve in a stable condition.
The term "labile reagent" is used herein to include any reagent that is susceptible to alteration or degradation when stored in aqueous solution at ambient temperature. Examples of labile reagents include: biomolecules, such as proteins, peptides, or nucleic acids, or derivatives thereof, or chemicals such as enzyme cofactors, enzyme substrates, nucleotide triphosphates (ribo- or deoxyribo-nucleotide triphosphates), or salts. Examples of proteins include enzymes (for example polymerases, such as DNA or RNA polymerases), and antibodies (native or recombinant, and fragments or derivatives thereof that retain antigen binding activity). An antibody (or fragment or derivative) may be present in the formulation in the absence of an antigen bound by the antibody. Examples of nucleic acids include DNA, RNA, nucleic acid primers, and carrier nucleic acid.
The labile reagent may be an amplification reagent for amplifying a target nucleic acid (for example, by a self-sustained amplification reaction), or a detection reagent for detecting product resulting from amplification of target nucleic acid.
The amplification reagent may be any reagent required for amplification of a target nucleic acid. For example the amplification reagent may comprise an enzyme activity, or a primer. The enzyme activity may, for example, be a DNA or RNA polymerase, such as an RNA-dependent DNA polymerase, a DNA-dependent DNA polymerase, a DNA/RNA duplex-specific ribonuclease, or a DNA-dependent RNA polymerase.
In an embodiment of the invention, a lyophilisation formulation of the invention comprises a polysaccharide, a low molecular weight saccharide, and enzyme activities required for self-sustained amplification of a target nucleic acid (for example, using a method of the invention) in the absence of enzyme cofactor(s) (for example magnesium ions), primers, rNTPs, and dNTPs required for specific amplification of the target nucleic acid.
The enzyme activities may be RNA-dependent DNA polymerase, DNA-dependent DNA polymerase, DNA/RNA duplex-specific ribonuclease, and DNA-dependent RNA polymerase enzyme activities.
It will be appreciated that methods of the invention may be carried out in the presence of a polysaccharide and a low molecular weight saccharide (for example, resulting from provision
of reagents required for amplification of the target nucleic acid with a lyophilised formulation of the invention).
Examples of suitable polysaccharides are starch, a dextran, or a derivative of a dextran (for example dextran sulphate). The molecular weight of the polysaccharide is typically in the range from about 10-200KD, usually 50-100kD. The polysaccharide may be linear or branched.
The low molecular weight saccharide may be a monosaccharide, disaccharide, or trisaccharide. Examples of suitable disaccharides include trehalose, sucrose, and maltose. Rehydration speeds of formulations of the invention that include trehalose have been found to be particularly fast. Inclusion of trehalose in lyophilised formulations of the invention that comprise an enzyme has been found to maintain the activity of the enzyme for long periods when stored at temperatures up to 37°C.
The low molecular weight saccharide may be present in an amount from 2.5-15% (w/v) of the formulation.
The polysaccharide and low molecular weight saccharide may be present in a total amount of 4-12% (w/v) of the formulation.
A lyophilisation formulation of the invention may further comprise an inert protein such as BSA or casein. BSA can readily be obtained as an RNase-free preparation.
A lyophilisation formulation of the invention may further comprise a sugar-alcohol.
There is also provided according to the invention a lyophilisation formulation of the invention which has been lyophilised (referred to as a lyophilised formulation of the invention). Methods of lyophilisation are known to those of ordinary skill in the art. A suitable method is described in Example 3 below.
Lyophilised formulations of the invention can maintain labile reagents in a stable condition at temperatures of 37°C for at least a year (Figures 5 and 6 show that 100% signal strength is obtained after storage at 37°C for one year). In contrast, lyophilised formulations described in US 5,556,771 show loss in activity after storage at 35°C for 61 days (Table 4 of US 5,556,771 shows that the average relative light units (RLU) for "Reagents wim DNA target" at 61 days is 90.9% of me average RLU at 0 days). The instructions provided with the SmartMix™ HM
of Cepheid (a kit comprising lyophilised reagents for PCR amplifications) specifies that the lyophilised reagents must be stored at 2-8°C. The instructions provided with the commercially available Nuclisens® Basic Kit Amplification Reagents of Biomerieux (a kit containing lyophilised reagents for NASBA-based nucleic acid amplification) specify that the amplification reagents should be stored at <-20°C. The instructions provided with the commercially available GEN-PROBE® AFITMA® General Purpose Reagents (GPR) 250 Kit (comprising lyophilised reagents for carrying out transcription-mediated amplification) specifies that the lyophilised reagents should be stored at 2-8°C. Lyophilised formulations of the invention do not need to be refrigerated.
The Applicant has surprisingly found that lyophilised formulations of the invention that comprise an enzyme (for example, an enzyme or combination of enzymes required to carry out a method of the invention) are able to maintain the enzyme(s) in a stable condition even in the absence of (or with little, e.g.